ABSTRACT The A20 ubiquitin-editing enzyme is a key negative regulator of NF-?B and innate immune signaling pathways. A20 contains seven zinc finger (ZF1-7) domains, of which ZF4 interacts with lysine 63 (K63)-linked polyubiquitin chains to suppress NF-?B signaling. Including A20, there are 10 genes containing A20 ZF domains encoded in the human genome; however, with the exception of A20 the functions of these proteins are poorly understood. ZFAND6 (also known as AWP1) contains an A20-like ZF domain that shares significant sequence homology with A20 ZF4, and also harbors an AN1-type ZF. Although published overexpression studies suggest that ZFAND6 may inhibit NF-?B, the specific pathways regulated by ZFAND6 and its physiological roles have remained elusive. In preliminary studies for this exploratory proposal, we have used gene targeting to generate mice lacking the Zfand6 gene. Zfand6?/? mice are born at expected Mendelian frequencies with no obvious developmental or immune abnormalities. RNA sequencing of Zfand6?/? bone marrow-derived macrophages revealed spontaneous induction of interferon-stimulated genes (ISGs), which rendered these cells highly resistant to infection with a broad range of DNA and RNA viruses. The DNA sensing pathway adaptor STING and the antiviral transcription factor IRF1 were both upregulated in Zfand6?/? cells, which may drive the resistance of ZFAND6-deficient cells to virus infection. ISGs were upregulated in the spleen, but not in the lungs, of Zfand6?/? mice, and knockout mice were more susceptible than wild-type mice to mortality upon challenge with a low dose of influenza A virus (IAV) despite comparable viral loads. Therefore, loss of ZFAND6 may perturb immune homeostasis and exacerbate virus-induced inflammation and immunopathology. The central hypothesis driving these investigations is that ZFAND6 targets key innate immune signaling proteins for degradation to prevent the spontaneous induction of ISGs which may contribute to overexuberant inflammation triggered by virus infection. To address this hypothesis we will perform the following Specific Aims: 1) Determine the mechanisms of ZFAND6 inhibition of antiviral signaling, and 2) Determine the role of ZFAND6 in regulating the inflammatory response to IAV infection. Completion of these studies will define a physiological role for ZFAND6 in restricting innate antiviral immunity and expand our knowledge on the mechanisms of immune homeostasis which serve to mitigate virus-induced inflammation.